Structure, Function and Antigenicity of Coronavirus Spike Proteins

Coronaviruses have the largest genomes among known RNA viruses and are phylogenetically divided into fourgenera. Some betacoronaviruses, such as HKU1, circulate annually in humans and cause mild yet prevalentrespiratory disease whereas others, such as SARS-CoV and the recently emerged MERS-CoV, have causedpandemics with high case-fatality rates. Due to their pandemic potential and airborne transmissibility, highlypathogenic coronaviruses are now classified as NIAID Category C priority pathogens. Coronavirus cell tropismand host range are in large part determined by the viral surface spike (S) glycoprotein, which is the largestknown class I viral fusion protein. After binding to host receptors and activation by host proteases, the Sproteins undergo large conformational rearrangements that result in fusion of the viral and host-cellmembranes. A molecular understanding of the structure, function and antigenicity of intact, trimeric S proteinswould identify sites of vulnerability that could be targeted by vaccines, therapeutic antibodies and small-molecule antivirals. However, structural studies have been primarily limited to small S protein fragments, whichhas precluded a unifying structural framework for the biology of coronavirus S proteins. To address this knowledge gap, we have generated soluble, trimeric S proteins from HKU1 and MERS-CoV that are amenable to structural analysis by X-ray crystallography and cryo-electron microscopy. We willdetermine atomic-level structures of these S proteins in both the prefusion and postfusion conformations,which will identify commonalities and differences among divergent betacoronaviruses and define theconformational end-states of the fusion process (Aim 1). With these constructs and a range of biochemical andbiophysical assays, we will determine the molecular basis for receptor-induced conformational changes andinvestigate the effects of host proteases and acidification on this process (Aim 2). The combination of thesestudies will provide key molecular insights into S protein-mediated membrane fusion and answer long-standingquestions regarding S protein triggering. Similar to other class I fusion proteins, such as influenzahemagglutinin (HA) and HIV-1 envelope (Env), coronavirus S proteins are the primary target for neutralizingantibodies and are thus a critical component of developmental vaccines. Currently, the best-characterizedantibodies against coronaviruses target the receptor-binding domain (RBD) of the S protein and preventbinding to host cells. The RBD, however, is the most variable part of the spike protein and antibodies thattarget this domain are unlikely to be cross-reactive, similar to most HA head-binding antibodies. Therefore, wewill define the epitopes and mechanisms of antibody-mediated neutralization for novel, non-RBD-directedneutralizing antibodies isolated by our collaborator Dr. Barney Graham (Aim 3). By identifying conserved sitesof vulnerability, these studies will provide the foundation for the development of immunotherapies and vaccinesthat broadly protect against highly pathogenic betacoronaviruses, including those that have yet to emerge.